Humans must breath nearly flawlessly from birth. At the same time, breathing must be both absolutely stable for the entire life of the organism as well as absolutely flexible on a breath by breath basis, to meet changing respiratory and behavioral demands such as arise from exercise, or speech. A number of disorders of human health are directly related to the brainstem networks underlying breathing such as apnea of prematurity and Sudden Infant Death Syndrome in infants or sleep apnea in adults. We hypothesize the mammalian respiratory rhythm is generated by the interaction of two respiratory oscillators that can be identified by the genes they express early in development. In this project we propose to use a combination of molecular genetics, electrophysiology, and anatomy to determine the physiological properties, behavioral necessity, gene expression, and developmental origins of medullary Dbx1 and Atoh1 neurons. Using intracellular recording in genetically fluorescently labeled slices of mouse brainstem, we will determine whether the physiological properties of genetically defined neurons correspond to their hypothesized function in generating breathing. We will determine the ability to respond to changes in pH or to the activation of opioid and/or somatostatin receptors is limited to specific genetic lineages. We will use three complementary genetic approaches to determine the necessity of genetically defined brainstem neurons in generating respiratory output. Using anatomical analysis, we will determine whether developmentally defined ventrolateral medulla (VLM) neurons express the neurokinin 1 receptor, somatostatin peptide, parvalbumin, and or the somatostatin 2a receptor. We will determine whether these populations are glutamatergic by combining immunohistochemistry and in situ hybridization in wild-type and GFP expressing transgenic reporter lines. We will determine the anatomical effects of loss of Atoh1 or Dbx1 genes. We will determine how the genome encodes specific populations of brainstem neurons. We will describe the global brainstem expression of Atoh1 and Dbx1 derived neurons by generating high-resolution digital atlases of neonate and adult brainstem. PUBLIC HEALTH RELEVANCE: A number of disorders of human health are directly related to the brainstem networks underlying breathing such as apnea of prematurity and Sudden Infant Death Syndrome in infants or sleep apnea in adults. Understanding the genetic organization of the brainstem neurons that generate breathing is essential for the diagnosis and treatment of breathing disorders.